In mammals the c-erbAalpha gene encodes two overlapping mRNAs which are identical except for their 3' terminal exons. The protein product of one of these mRNAs is the alpha-thyroid hormone receptor (TRalpha1), a nuclear receptor protein which mediates the cellular response to thyroid hormone. The other mRNA encodes a non-hormone binding variant (TRalpha2) which functions as a dominant negative repressor, antagonizing the function of TRalpha1. The ratio of receptors and their corresponding mRNAs varies in a tissue-specific and stage- specific manner. Alternative processing of the erbAalpha mRNA thus determines the cellular response to thyroid hormone. A remarkable feature of this locus is that it partially overlaps the 3' end of a gene for another receptor protein, Rev-ErbAalpha (RevErb), which is transcribed in the opposite direction. In most cells, the ratio of TRalpha1 to TRalpha2 mRNAs varies with the level of RevErb expression. Since the 3' end of RevErb overlaps the 3' exon of TRalpha2 but not TRalpha1, it has been hypothesized that base pairing interactions between RevErb and TRalpha2 transcripts selectively block expression of TRalpha2 relative to TRalpha1. Further evidence suggests that RevErb mRNA specifically blocks splicing of the TRalpha2-specific 3' exon. Research proposed here will investigate molecular mechanisms which regulate alternative processing of TRalpha1 and TRalpha2 mRNA. The specific aims of the research are as follows: (1) To characterize cis-acting elements which regulate mRNA processing. These experiments will focus on the function of two intronic splicing enhancers specific for processing TRalpha2 mRNA. These elements will be further mapped and proteins which bind to these elements will be characterized. (2) To characterize the role of RevErb expression in altering the ratio of TRalpha1 and TRalpha2. Cells stably transfected with minigenes expressing RevErb mRNA under the control of a regulated promoter will be constructed to examine the effect of RevErb on expression of TRalpha1 and TRalpha2 mRNAs. The extent of overlap between RevErb and TRalpha1 will be explored by constructing mutations within the region of overlap. (3) To determine whether the complementary transcripts form a stably base-paired duplex. A sensitive PCR- based strategy will be used to search for duplexes between spliced and unspliced RNAs. RNase protection assays will be used as an alternative approach. The proposed research is important for understanding more fully regulation of the physiological response to thyroid hormone in mammals and requirements fir for base-paired antisense interactions between complementary transcripts in vivo.